Cyclosporins are a group of biologically active fungal metabolites. The major components, cyclosporin A and C are non-polar cyclic oligopeptides with immunosuppressive, antifungal and antiphlogistic activity. Cyclosporin A is used frequently as a immunosuppressive agent in organ transplantation. Cyclosporin A is also under investigation for suppression of graft versus host disease in bone marrow transplants and studies have indicated its usefulness in treating diabetes mellitus (type 1). Stiller, et al. Science, 223: 1362-1367 (1984). A number of reports have proven that cyclosporin A is better than standard immunosuppressive drugs azathioprine and prednisone in maintaining the viability of heterotropic cadaveric renal, liver, lung, heart and pancreas transplants. European Multicentre Trial, Lancet, 2: 57-60 (1982).
While cyclosporin A is an extremely useful immunosuppressive agent potentially harmful side effects do exist. One harmful side effect which has been reported is nephrotoxicity. Merion et al., New England Journal of Medicine 310: 148-154 (1984); Rosenthal et al., Surg. Gynecol. Obstet. 157: 309-315 (1983). Delivery of appropriate dosages of cyclosporin A is also a problem. It has been reported that following oral administration, the bioavailability of cyclosporin A is very poor. Beveridge et al., Curr. Ther. Res., 30: 5-18 (1981). Also, inter-subject variability is very high in the case of oral administration of cyclosporin A. Moyer et al., Clin. Biochem. 19: 83-89 (1986). In view of the drawbacks associated with oral administration of cyclosporin A, intravenous administration is the preferred route. However, the available intravenous formulation has been withdrawn from the market due to the toxicity of the carrier, cremophore. Development of an alternative intravenous dosage form of cyclosporin A is therefore needed.
It has been known that phospholipids under appropriate conditions can spontaneously reform, in the presence of water, into closed membrane systems. Electron microscopy reveals that these structures are made of a number of concentric bilayers of phospholipid molecules, and are called liposomes. The usefulness of liposomes as a membrane model system arises from the fact that, as the dry phospholipids undergo their sequence of molecular rearrangements, there is an opportunity for an unrestricted entry of hydrophilic solutes between the planes of hydrophilic head groups (aqueous compartments). Similarly, sequestration of hydrophobic solutes occurs within the hydrophobic bilayers. The result is the production of a delivery system that can contain varying amounts of the drug depending on the type of interaction between the solute and the phospholipid.
Many methods have been proposed for the preparation of liposomes, the first and most widely used being the film method. Briefly, lipids of the desired composition in a solution with an organic solvent are dried in the form of a thin film on the walls of a roundbottom flask. A hydrophobic drug can be included in the film at this stage. The dry film is hydrated by adding a suitable aqueous phase and gently swirling the flask. With a hydrophilic compound, an aqueous solution of it is used for hydration. The liposomes formed by this procedure generally have a number of concentric bilayers and are called multilamellar vesicles.
Liposomes have been evaluated as potential drug delivery systems to introduce biologically active material into cells. Poznansky and Juliano, Pharmacol. Rev., 36:277-336 (1984); Ryman, B.E. and Tyrrell, D.A. 1980: Liposomes-Bags of Potential, Essays in Biochemistry 16: P.N. Campbell and R.D. Marshall, Academic Press, London pp. 49-98. Several routes of administration have been tried for the administration of liposomes, for example, intravenous, subcutaneous, intraperitoneal, and oral delivery. Gregoriadis and Allison, Ed. "Liposomes in Biological Systems", John Wiley & Sons, New York pp. 153-178 (1980) An important advantage with liposomal drug delivery is the change in tissue distribution and binding properties as compared to the free forms of the drug resulting in enhanced therapeutic index and decreased toxicity. Examples include decreased nephrotoxicity of cyclosporin A [(Hsieh et al., Transplantation Proceedings, Vol. XVII:1397-1400 (1985)], and reduced cardiotoxicity and nephrotoxicity of doxorubicin and cisplatin, respectively as compared to the free forms of the drugs. Rahman et al., Cancer Res., 42:1817-1825 (1982); Forssen and Tokes, Cancer Res., 43:546-550 (1983).
Historically liposomes have been studied as suspensions and only recently freeze-dried into a powder form to enable redispersion at the time of administration. Gorden et al., Drug Dev. Ind. Pharm., 8:465-473 (1982); Crommelin and VanBommel, Pharm. Res. 1:159-164 (1984); Evans et al., U.S. Pat. Nos. 4,311,712 and 4,370,349. A systematic optimization of freeze-drying of liposomes has been done in the presence of various cryoprotectants using carboxyfluorescein as the marker Fransen et al., Int. J. Pharm. 33:27-35 (1986).
Liposomal instability has been a major concern for long-term storage. Several changes such as change in size distribution, drug content and sedimentation can occur upon storage for an extended period. Therefore, providing liposomes in a dry powder form that is readily redispersible is highly desirable. While freeze-drying has been employed to make dry powder liposome and drug mixtures, researchers have reported problems of leakage of the drug upon reconstitution. In some cases, liposomes have been stabilized using adjuvants such as sugars to maintain the integrity of liposomal membranes during freeze-drying. Strauss & Hauser, Proc. Natl. Acad. Sci. USA, Volume 83:2422-2426 (April 1986); Strauss et al., Biochimica et Biophysica Acta 858:169-180 (1986). In the Evans patents reconstituted preparations of liposomes having steroid encapsulated therein exhibited 28% loss of the amount of steroid present in the liposome preparation prior to freeze-drying. In another instance, the Evans patents report that liposomes formed after freeze-drying retained 28% of the Angiotensin II present in liposomes prior to freeze-drying. Accordingly, there is a need for a freeze-dried liposome composition containing cyclosporin which when reconstituted in an aqueous media yields liposomes which incorporate substantially all of the cyclosporin present in the composition prior to freeze-drying.